Mice transgenic for the human myotonic dystrophy region with expanded CTG repeats display muscular and brain abnormalities.

The autosomal dominant mutation causing myotonic dystrophy (DM1) is a CTG repeat expansion in the 3'-UTR of the DM protein kinase (DMPK) gene. This multisystemic disorder includes myotonia, progressive weakness and wasting of skeletal muscle and extramuscular symptoms such as cataracts, testicular atrophy, endocrine and cognitive dysfunction. The mechanisms underlying its pathogenesis are complex. Recent reports have revealed that DMPK gene haploinsufficiency may account for cardiac conduction defects whereas cataracts may be due to haploinsufficiency of the neighboring gene, the DM-associated homeobox protein (DMAHP or SIX5) gene. Furthermore, mice expressing the CUG expansion in an unrelated mRNA develop myotonia and myopathy, consistent with an RNA gain of function. We demonstrated that transgenic mice carrying the CTG expansion in its human DM1 context (>45 kb) and producing abnormal DMPK mRNA with at least 300 CUG repeats, displayed clinical, histological, molecular and electrophysiological abnormalities in skeletal muscle consistent with those observed in DM1 patients. Like DM1 patients, these transgenic mice show abnormal tau expression in the brain. These results provide further evidence for the RNA trans-dominant effect of the CUG expansion, not only in muscle, but also in brain.

The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

In-vivo delivery of therapeutic proteins by genetically-modified cells: comparison of organoids and human serum albumin alginate-coated beads.

We have designed a self-assembling multimeric soluble CD4 molecule by inserting the C-terminal fragment of the alpha chain of human C4-binding protein (C4bp alpha) at the C-terminal end of human soluble CD4 genes. This CD4-C4bp alpha fusion protein (sMulti-CD4) and two other reference molecules, a fusion protein of human serum albumin (HSA) and the first two domains of CD4 (HSA-CD4) and monomeric soluble CD4 (sMono-CD4), were delivered in vivo by genetically modified 293 cells. These cells were implanted in mice as organoids and also encapsulated in HSA alginate-coated beads. sMulti-CD4 showed an apparent molecular weight of about 300-350 kDa, in accordance with a possible heptamer formula. sMulti-CD4 produced either in cell culture or in vivo in mice appeared to be a better invitro inhibitor of HIV infection than sMono-CD4. Plasma levels of sMulti-CD4, HSA-CD4, and sMono-CD4 reached approximately 2,300, 2,700, and 170 ng/mL, respectively, 13 weeks after in-vivo organoid implantation, which had formed tumours at that time. This suggests that the plasma half-life of sMulti-CD4 is much longer than that of sMono-CD4. The 293 xenogeneic cells encapsulated in HSA alginate-coated beads remained alive and kept secreting sMono-CD4 or HSA-CD4 continuously at significant levels for 18 weeks in nude mice, without tumour formation. When implanted in immunocompetent Balb/c mice, they were rejected two to three weeks after implantation. In contrast, encapsulated BL4 hybridoma cells remained alive and kept secreting BL4 anti-CD4 mAb for at least four weeks in Balb/c mice. These results suggest the clinical potential of the C4bp-multimerizing system, which could improve both the biological activity and the poor in-vivo pharmacokinetic performance of a monomeric functional protein like soluble CD4. These data also show that a systemic delivery of therapeutic proteins, including immunoglobulins, can be obtained by the in-vivo implantation of engineered allogeneic cells encapsulated in HSA alginate-coated beads.

Use of herpes simplex virus thymidine kinase to improve the antiviral activity of zidovudine.

Many antiviral drugs must be metabolized to their active form by cellular enzymes. Their antiviral activity may therefore be limited by an inefficient metabolism, leading to low intracellular concentration of the active form or to the accumulation of toxic intermediate metabolites. Gene transfer might be used to overcome such limitations by transducing a gene able to increase intracellular drug metabolism. To prove such a concept, we chose the well-studied paradigm of zidovudine (AZT) metabolism and anti-HIV activity. AZT-triphosphate is the active form of AZT, acting through inhibition of HIV reverse transcription. In human cells, the rate-limiting step for AZT phosphorylation is catalyzed by the thymidylate kinase. We thus tested the capacity of herpes simplex virus type 1 thymidine kinase, which possesses a thymidylate kinase activity, to improve AZT metabolism and antiviral activity. Our results show enhanced AZT phosphorylation in HSV-1 TK-expressing lymphoid and monoblastoid cells, which correlated with significantly improved antiviral activity against different strains of HIV-1. The antiviral activity of Foscarnet, another reverse transcriptase inhibitor that does not require phosphorylation, remained unchanged. These results suggest that gene transfer might be envisioned for genetic pharmacomodulation of antiviral drugs.

Expression of a Tat-inducible herpes simplex virus-thymidine kinase gene protects acyclovir-treated CD4 cells from HIV-1 spread by conditional suicide and inhibition of reverse transcription.

Cellular expression of the herpes simplex virus type 1 thymidine kinase (HSV1-TK) gene promotes cell death in the presence of specific nucleoside analog substrates such as acyclovir (ACV). We have reported that lymphoid CD4+ cells harboring an HSV1-TK gene, under the transcriptional control of the HIV-1 long terminal repeat (HUT-TK), are completely protected from HIV-1 spread in the presence of 10 microM ACV. In this report we clarify the efficiency, generality, and mechanism of this protective effect. We show that the protection from HIV-1 spread in HUT-TK cells obtains from both an inhibition of HIV reverse transcription by ACV metabolites and an HIV-induced and ACV-dependent cell killing. We also demonstrate that monocytic cells harboring the HIV-1-inducible HSV1-TK gene are protected from HIV spread in the presence of ACV. These observations facilitate the design of therapeutic strategies to limit HIV replication based on HSV1-TK expression.

Loss of CD4 membrane expression and CD4 mRNA during acute human immunodeficiency virus replication.

Using mAbs and genomic probe to the CD4 molecule, the HIV receptor, we demonstrated that HIV replication induces the disappearance of its functional receptor from the cell surface by two distinct mechanisms. First, after being expressed onto the cell surface, HIV envelope gp110 will complex CD4, efficiently masking the CD4 epitope used by the virus to bind its receptor. This phenomenon occurs on the surface of each infected cell and is not due to the release of soluble gp110; infection with recombinant HIV/vaccinia viruses expressing a mutated HIV env gene designed to prevent gp110 release from the cell surface induces a similar gp/CD4 complexes formation. Second, virus replication induces a dramatic and rapid loss of CD4 mRNA transcripts, preventing new CD4 molecules from being synthesized. These two mechanisms of receptor modulation could have been developed to avoid reinfection of cells replicating the virus as well as to produce more infectious particles. These results suggest that the classical virus interference documented for other retroviruses might not only be due to receptor/envelope interaction, but might also depend on receptor gene expression.

[Functional inhibition by cyclosporin A of the lymphocyte receptor for the AIDS virus (HIV)]. / Inhibition fonctionnelle par la ciclosporine A du récepteur lymphocytaire du virus du sida (HIV).

The Human Immunodeficiency Virus (HIV) displays a selective tropism for cells expressing the CD4 molecule which, by itself, represents at least part of the specific receptor for this virus. However, modification of the activation state of each individual cell seems critical not only for virus replication but also for its binding and subsequent penetration into its target. We demonstrate here that Cyclosporin-A (CSA), a drug which inhibits IL-2 dependent T-lymphocyte proliferation and differentiation and which is known for its immunosuppressive activity, can prevent subsequent virus binding to cells otherwise susceptible to HIV. Normal T-lymphocytes were preincubated in vitro with CSA at concentrations that were in the same range than those reached in the serum of treated patients. This resulted in the complete disappearance of HIV receptors (HIV-R), as assessed by the direct measure of specific binding of fluoresceinated HIV (HIV-FITC), and in the subsequent inhibition of HIV replication in cultured cells. Moreover CSA pretreatment of IL-2 independent transformed cells derived from the CEM line, before their infection, strongly inhibited HIV adsorption as well as further virus replication. These results provide a new experimental basis for the potential application of CSA in the treatment of HIV-related diseases.

Is there correlation of T cell proliferative functions and surface marker phenotypes in patients with acquired immune deficiency syndrome or lymphadenopathy syndrome?

We have investigated the respective role of quantitative T lymphocyte subset abnormalities, interleukin-2 (IL-2) production and responsiveness to IL-2, in the proliferative deficiency that is observed in acquired immune deficiency syndrome (AIDS) and Lymphadenopathy syndrome (LAS) patients or even in some apparently healthy male homosexuals. 83 subjects were evaluated: 35 symptom free male homosexuals (HC), 24 LAS and 24 AIDS patients. As expected, many HC and most patients presented with T lymphocyte subset imbalance. These quantitative defects were associated with decreased reactivity to PHA and reduced production of IL-2 by PHA stimulated lymphocytes. No correlation however could be found between these two functions and variations in the T lymphocyte subset distribution. On the other hand, PHA responsiveness appeared to closely depend on IL-2 activity. Addition of exogenous IL-2 to lymphocytes from patients with low proliferative responses, stimulated with suboptimal PHA concentration, enhanced proliferation in some but not all the cases. In most instances this increase never reached the levels observed with similarly treated cells from normal individuals. In these patients, the limited number of lymphocytes which express IL-2 receptors upon PHA stimulation may explain both low PHA reactivity and reduced IL-2 responsiveness. These data indicate some possible mechanisms of immunodeficiency in AIDS and LAS.

Mixed lymphocyte culture suppressor cells can be induced by non-HLA differences in man.

Lymphocytes (A) sensitized in vitro by cells from a HLA-identical sibling (B) for 8 days showed inhibiting effects when added to fresh mixed lymphocyte cultures (MLC) where A responders were stimulated by cells from other family members in a ratio of 1:1:1. In 23 of 31 such pairs tested in 15 families, proliferative activities in these 6-day second-step MLC were inhibited by 54 +/- 18% in the presence of A'B sensitized cells as compared to control cultures with modulating A' cells similarly preincubated but in the absence of B stimulators. In addition, A'B could also suppress MLC responses of B in 12 of the 17 pairs in which this was tested. Inhibition was not due to cytotoxic elimination of stimulators and it was radiation sensitive. Suppression appeared to be specific but it did not seem to be restricted by HLA-A, -B, or -DR determinants. Hence, these results indicate that suppressor cells generated after priming by HLA-identical cells can regulate allogeneic proliferative responses even when they are directed to HLA differences.

[Acquired immunodeficiency syndrome and related syndromes. Critical analysis of biological tests of cell-mediated immunity]. / Syndrome d'immunodéficit acquis et syndromes apparentés. Analyse critique des examens biologiques de l'immunité à médiation cellulaire.

In order to assess the actual value of the in vitro tests of cellular immunity performed to provide diagnostic evidence for acquired immune deficiency syndrome (AIDS) or related syndromes, we have evaluated T-lymphocyte subsets and in vitro proliferative responses. The respective profiles of 44 symptom-free homosexual men, 30 patients with AIDS and 42 patients with lymphadenopathy were compared with each other and with profiles of blood bank volunteers, using stepwise discriminant analysis. At first investigation, control homosexuals, AIDS patients and lymphadenopathy patients displayed varying degrees of the same type of immunological abnormalities. The percentage of OKT4+ cells was the most discriminant variable between blood donors and control homosexuals, AIDS or lymphadenopathy patients. The latter differed from homosexuals by a higher percent of OKT8+ cells and from AIDS patients by a lesser reduction in T-lymphocyte counts. OKT4+ cell counts differentiated AIDS patients from control homosexuals. However, none of these major discriminant variables could classify correctly more than 70% of subjects in the homosexual and in the two patient groups. Thus, a single investigation of T-lymphocyte immune parameters does not provide substantial diagnostic and prognostic evidence.

Critical analysis of T cell subset and function evaluation in patients with persistent generalized lymphadenopathy in groups at risk for AIDS.

In order to determine whether there exists a distinct immunological profile that discriminates between healthy homosexual men and those expressing persistent generalized hyperplastic lympadenopathy GHL and which of the observed abnormalities in GHL would be most indicative of patients who would eventually develop AIDS, we evaluated T lymphocyte subset and in vitro proliferative responses. The respective profiles of 44 symptom free homosexual men (HC), 42 patients with GHL and 30 cases of AIDS were compared to that of blood bank volunteers (BBV) and to each other, using stepwise discrimination analysis. HC, GHL and AIDS patients demonstrated upon their first investigation varying degrees of the same type of abnormal immunological characteristics. The percentage of OKT4+ cells was the most discriminant variable between BBV and HC, GHL or AIDS. GHL was distinguished from HC primarily by a higher percentage of OKT8+ cells and from AIDS by higher T lymphocyte counts, while the OKT4+ cell count discriminated between AIDS and HC. However, none of these major discriminant variables could classify correctly more than 71% within the HC and the two patient groups. Thus, there exists a continuity between GHL and AIDS in the range of observed immunological abnormalities without clearcut boundaries. This, together with the existence of abnormal background values in HC will not permit a single investigation of T lymphocyte immune parameters to provide substantial diagnostic and prognostic evidence.

[Allogeneic immune response after blood transfusion in patients awaiting renal transplantation]. / Réponse immune allogénique après transfusion sanguine chez des malades en attente de transplantation rénale.

To investigate the mechanisms of the beneficial effect of blood transfusions (BT) on subsequent kidney transplant survival, we studied the influence of 3 planned BT on lymphocyte reactivity and on lymphocytotoxic antibody (LT) production in previously non-transfused uremic patients. A sustained and non-specific decrease in mixed lymphocyte reactivity (MLR) was observed in approximately 60% of the cases, whereas other patients had only a transient decrease but otherwise normal or even increased responses. Neither the pre-BT degree of immune responsiveness nor the clinical status of the patients had any influence on this phenomenon. Similarly, hepatitis B seroprophylaxy or vaccination did not interfere with this BT effect. Anti-HLA, LT, which are potentially harmful for the transplant, were noted in 13% of cases, while 13% additional cases displayed "cold" anti-B lymphocyte LT which do not have the same prognostic value. In some instances, suppression of cellular reactivity developed concurrently with LT production, which indicates that there is no interaction between cellular and humoral responses induced by BT.

Interleukin-2 production after blood transfusions in prospective kidney transplant recipients.

We have investigated whether suppression of the allogeneic response after blood transfusion (BT) could be due to inhibition of the production or activity of Interleukin-2 (IL-2), a soluble mediator involved in T lymphocyte proliferation. Reduction of IL-2 production after BT was less frequent than, but significantly associated with, non specific MLR suppression. However, MLR suppressor cells did not inhibit the release of IL-2 from autologous pre-BT lymphocytes. In addition, post-BT soluble suppressor factors of the MLR did not affect the ability of IL-2 to promote cell proliferation. Thus, although MLR suppression after BT is associated with reduced IL-2 production, this is not a major mechanism involved in this effect.

The influence of blood transfusions on lymphocyte reactivity in man.

The effect of planned blood transfusion (BT) on lymphocyte reactivity in previously non-transfused uraemic patients has been investigated. A sustained and non-specific decrease in MLR was observed in approximately 60 per cent of the cases. Other patients had only a transient decrease, normal or increased response. Lymphocyte suspensions whose proliferation was reduced after BT suppressed the response of autologous cells taken before BT. Neither pre-BT degree of immune responsiveness nor clinical status of the patients had any influence on this phenomenon.

Monocytotoxic antibodies after bone marrow transplantation in aplastic anemia.

Pre- and post transplant sera from 51 cases of bone marrow transplants performed for severe aplastic anemia were tested on monocytes (M) and corresponding B cells (B) from a panel of unrelated donors. One-third of the sera were cytotoxic for B and M either from different or from the same individuals, while 45% reacted only to M and appeared to recognize non-HLA M-associated antigens. No significant reaction to endothelial cells was obtained from these sera. The subsequent clinical course was not associated with any reaction pattern of pre-transplant sera. There was a significant relationship between rejection and the development of M antibodies after grafting, but since these were also found in many other patients without rejection, their occurrence has no predictive value for an individual patient.

A longitudinal study of in vitro tests for lymphocyte function in rheumatoid arthritis.

In vitro tests of lymphocyte function have been performed in 61 patients with ;classical' or ;definite' rheumatoid arthritis. In vitro lymphocyte function was assessed by lymphocyte transformation responses to phytohaemagglutinin (PHA), Pokeweed mitogen (PWM), Candida antigen, and herpes simplex type I (HSV1). Follow up data were available after 6 months of treatment in 32 of these patients. Spontaneous lymphocyte transformation was assessed in all patients. Results obtained in patients with rheumatoid arthiritis were compared to those seen in a normal control population. Disease activity of patients with rheumatoid arthritis was assessed using standard clinical methods. Lymphocytes from patients with rheumatoid arthritis showed a similar degree of spontaneous transformation to that seen in normal subjects. In contrast, lymphocytes from patients with rheumatoid arthritis responded less well to PHA and Candida and HSV1 antigens when compared to normal patients. In patients with rheumatoid arthritis the response to PWM was markedly enhanced compared to normals. Clinical improvement was noted in 19 of the 32 patients seen at follow up, all of whom had received gold or penicillamine therapy. The abnormal responses of PHA and PWM seen before treatment became normal in those patients who improved clinically. The responses to Candida and HSV1 antigens not only returned to normal following treatment but were increased above those seen in normal controls. A statistically significant association was seen between clinical improvement and improvement of in vitro tests of lymphocyte function.

A double-blind, controlled trial of levamisole in the treatment of recurrent herpes labialis.

A double-blind, controlled trial of levamisole in the prevention and treatment of recurrent cold sores was performed. Forty-eight subjects received levamisole in a dosage of 2.5 mg/kg on two consecutive days each week for six months. The 51 control subjects were given a placebo identical to the drug in appearance. Both groups were given the same instructions. Nineteen subjects receiving levamisole and eight receiving placebo withdrew during the six months of the study. There were no significant differences between the levamisole-treated and control groups in the duration or severity of the lesions during the trial period or in the subjective assessment of drug efficacy by the participants at the end of the trial. Before treatment the frequency of lesions in the levamisole group was higher than in the control group. Only when this factor was taken into account by analysis of covariance did the decreased frequency of lesions during therapy appear significantly lower in the group receiving levamisole than in the placebo group. The difference remained clinically unimpressive. This study does not support earlier suggestions that levamisole, in these doses, is useful in the treatment of recrudescent circumoral herpesvirus infections.